Structured illumination microscopy (SIM) illuminates a specimen with structured or patterned light, which excites fluorescence in the specimen according to that pattern. A plurality of images are collected with the illumination shifted between each image collection. Analysis of a plurality of such images is used generate a super-resolution image.
Stimulated emission depletion (STED) microscopy is also a type of super-resolution microscopy, that operates by exciting fluorescence in a specimen, while de-exciting fluorophores to generate a super-resolution image.
Localization microscopy provides super-resolution by isolating emitters and fitting their images with the point spread function (PSF), thereby solving the problem that the width of the point spread function limits resolution. A sparse subset of emitters is activated at the same time, these emitters are localized precisely and they are then deactivated. The process is then repeated. with another subset. The collected photons (emitted, for example, during a fluorescent phase) are collected camera and the resulting image of the fluorophore is fitted. The process is repeated several thousand times so that all fluorophores can go through the bright state and be recorded. A computer is then used to reconstruct a super-resolved image.
Jerome Mertz (Dept. Biomedical Engineering, Boston University, MA) has described a method for phase contrast endoscopic imaging in thick biological tissues, termed Oblique Illumination Phase Contrast Endoscopy. This involves modifying an endoscope, such as that made by Muana Kea Technologies (trade mark) by adding two 1-mm wide LED illumination fibres to the central 2.8 mm optical fibre core detector. This configuration allowed Mertz to use Monte Carlo modelling to reveal a virtual oblique light source deep within the illuminated tissue. Subtraction of the left and right images generates a phase image. This technique was tested using 45-μm beads in agarose, and is said to have revealed good phase images at a depth of about 150 μm. Mertz also reports examining chick embryos and detecting red blood cells flowing in capillaries.
FIG. 1 shows a single sided scanning disc confocal microscope (see U.S. Pat. No. 4,927,254 to Kino and Xiao). Light from a broad area excitation source 1 is collimated by lens 2 as a beam 3 that is reflected by the dichroic layer of beamsplitter cube 4 and is converged by lens 5 to the surface of a spinning disk 6. This disk is perforated by a multitude of very small holes 7. The disc is spun rapidly on an axle 8. The light then passes through these holes indicated by beam 9 and is converged by lenses 10 and 11 to produce a series of focused spots in the specimen 12. Fluorescence generated at these spots returns back through the holes in the plate to the beam splitter. Since the fluorescence is of a longer wavelength than the excitation light it passes through the dichroic beamsplitter cube and is focused by lens 13 to form an image on the CCD chip 14. The signal from this chip forms a display image on the screen 15.
FIG. 2 is a schematic view of a Petran spinning disc confocal microscope (Petran, Hadraysky, Egger and Galambos, Tandem Scanning Reflected Light Microscope, Journal of the Optical Society of America, 58 (1968) pp. 661-664), also referred to as a tandem scanning Nipkow disc confocal. Light from light source 1 passes through lens 2 and is reflected by mirror 3 onto the surface of a spinning disk 4. The disk is perforated with a multitude of very small holes 5. Light passes through these holes and is collimated by lens 6, reflected by mirror 7 and passes through a dichroic beams splitter 8 the light then passes through lenses 9 and 10 of the microscope objective to form a focus within the specimen 11. Light generated within the specimen passes through the lenses and beam splitter and through inversion optics 12. These allow the lens 13 to produce a mirror image of the specimen. This mirror image passes through a matching set of holes 14 in the disc that are on the side of plate 180 degrees from the illumination holes. A lens 15 collects this light and brings it to a focus on a CCD 16 to form an image. During image observation the disc rotates around axis 17. The holes make up a few per cent of the disc area, and are also not truly in a Nipkow configuration as at least 100 holes are in the field of view at any one time.
FIG. 3 is a schematic view of a super resolution system as described by Stefan Hell using STED/RESOLFT/GSD principles. A TEMoo excitation wavelength light source 1 emits a beam of light which is focused by lens 2 and passed through a pinhole 3. The beam is collimated by lens 4 and passes as a Gaussian beam 5 and is reflected by beams splitter 62 scanning mirrors 7 and telecentric lenses 8 and 9 then passes through lens 10 which focuses it into the specimen 11. A de-excitation source 12 emits light 13 which passes through a phase filter 14 emerging as a doughnut mode beam 15. This beam is reflected from beam splitter cube 16 and is also reflected by mirror scanner 7 it also passes through the telecentric lenses 8 and 9 and is focused into the specimen 11 by lens 10. At the focus in the specimen light source 1 produces a Gaussian spot a magnified view of which is given in 17 the spot is surrounded by the doughnut mode image of the de excitation beam the cross section of which is shown in 18. This partly overlaps and erases the peripheral areas of the central spot. The resulting excited region remaining is shown in 19. Fluorescence from this spatially reduced region passes through the optical system and is focused by lens 20 through pinhole 21 and generates a signal in photomultiplier tube 22.
FIGS. 4A and 4B are reproductions of, respectively, FIGS. 10 and 11 of U.S. Pat. No. 7,755,063, entitled Super Resolution in Micro-Lithography and Fluorescence Microscopy to Stephen C. Baer. FIG. 4A shows a super resolution system operating by STED/RESOLFT/GSD in which the excitation light is delivered by one optical fibre and the de-excitation light is delivered via four or six individual discrete optical fibres whose tips surround the tip of the first fibre.
FIG. 5 is a reproduction of FIG. 12 of U.S. Pat. No. 7,755,063, and illustrates a system for photolithography of silicon wafers with ultrahigh resolution using a multiplicity of separate optical fibres to deliver the de activating light.
U.S. Pat. Nos. 7,864,314, 7,782,457, 7,710,563, 7,626,703, 7,626,695, 7,626,694 and 7,535,012 disclose systems for optical microscopy using phototransformable optical labels.